Plant genes that protect against herbivorous insects may be useful for heterologous expression in food and fiber crops (Boulter, “Genetic Engineering of Plants for Insect Resistance,” Outlook on Agriculture 18:2-6 (1989); Vogel et al., “Natural Proteinase Inhibitors,” Academic Press, New York (1968)). Proteinase inhibitors (PIs) are common in plants and have drawn attention as possible transgenes for insect defense in crops. PIs are of particular interest because they are generally the product of a single gene, and inhibit proteolytic enzymes of animal and fungal origin, but rarely plant origin, and therefore are thought to act as protective agents (Baldwin et al., “Rapid Changes in Tree Leaf Chemistry Induced by Damage: Evidence for Communication Between Plants,” Science 221:277-279 (1983); Brattsten, “Bioengineering of Crop Plants and Resistant Biotype Evolution in Insects: Counteracting Coevolution,” Archives of Insect Biochemistry and Physiology 17:253-267 (1991); Green et al., “Wound-Induced Proteinase Inhibitor in Plant Leaves: A Possible Defense Mechanism Against Insects,” Science 175:776-777 (1972); Hilder et al., “Transgenic Plants Conferring Insect Tolerance: Protease Inhibitor Approach,” in Kung, eds., Transpenic Plants, London: Academic Press, Inc., pp.317-338 (1993); Laskowski et al., “The Enzymes,” Academic Press, Inc., New York (1977)). Several studies have demonstrated that PIs might provide adequate protection against a variety of economically important lepidopteran insects (Broadway et al., “The Effect of Dietary Protein on the Growth and Digestive Physiology of Larval Heliocoverpa zea and Spodoptera exiqua,” J. Insect Physiol. 32:827-833 (1986); Hoy et al., “Feeding Response of Artogeia rapae (Lepidoptera:Pieridae) and Trichoplusia ni (Lepidoptera:Noctuidae) to Cabbage Leaf Age,” Environ. Entomol. 16:680-682 (1987); Johnson et al., “Expression of Proteinase Inhibitors I and II in Transgenic Tobacco Plants: Effect on Natural Defense Against Manduca sexta Larvae,” Proc. Natl. Acad. Sci. USA 86:9871-9875 (1989); Lipke et al., “Effect of Soybean Inhibitors on Growth of Tribolium confusum,” A Food Chem 2:410-414 (1954); Oppert et al., “Dietary Mixtures of Cysteine and Serine Proteinase Inhibitors Exhibit Synergistic Toxicity Toward the Red Flour Beetle, Tribolium Castaneum,” Comp. Biochem. Physiol. 105C:379-385 (1993); Sánchez-Serrano et al., “Wound-Induced Expression of a Potato Proteinase Inhibitor II Gene in Transgenic Tobacco Plants,” EMBO J. 6:303-306 (1987); Thomas et al., “Protease Inhibitors of Manduca Sexta Expressed in Transgenic Cotton,” Plant Cell Reports 14:758-762 (1995); Thomas et al., “Introduction and Expression of an Insect Proteinase Inhibitor in Alfalfa (Medicago sativa L.),” Plant Cell Reports 14:31-36 (1994); Xu et al., “Constitutive Expression of a Cowpea Trypsin Inhibitor Gene, CpTi, in Transgenic Rice Plants Confers Resistance to Two Major Rice Pests,” Molecular Breeding 2:167-173 (1996)).
The production and accumulation of PIs in plants can be activated by a variety of mechanisms. Potato, tomato and poplar PIs have been shown to be wound inducible, both at the site of wounding and systemically (Bradshaw et al., “Systemically Wound-Responsive Genes in Poplar Trees Encode Proteins Similar to Sweet Potato Sporamins and Legume Kunitz Trypsin Inhibitor,” Plant Mol. Biol. 14:51-59 (1990); Graham et al., “Regulation of Synthesis of Proteinase Inhibitors I and II mRNAs in Leaves of Wounded Tomato Plants,” Planta 169:399-405 (1986); Sánchez -Serrano et al., “Nucleotide Sequence of Proteinase Inhibitor II Encoding cDNA of Potato (Solanum Tuberosum) and Its Mode of Expression,” Mol. Gen. Genet. 203:15-20 (1986)). In contrast, the production of PIs in cabbage (Brassica oleracea), especially trypsin and chymotrypsin inhibitors, are linked to plant development (Broadway et al., “Regulatory Mechanisms of Tryptic Inhibitory Activity in Cabbage Plants,” Phytochem. 29:3721-3725 (1990)). Low levels of PI activity in cabbage are produced in young foliage in seedlings (Broadway et al., “Regulatory Mechanisms of Tryptic Inhibitory Activity in Cabbage Plants,” Phytochem. 29:3721-3725 (1990); Broadway et al., “Influence of Cabbage Proteinase Inhibitors in situ on the Growth of Larval Trichoplusia ni and Pieris rapae,” J. Chemical Ecology 18:1009-1023 (1992). When the plant reaches the 11-13 leaf stage, the level of PI activity gradually increases in young leaves, and reaches a maximal level of activity in the young foliage on mature plants. The production of PIs in cabbage is synchronized with the appearance of herbivorous insects in the field. Thus, the PIs are present when the resistance factor is most needed against these pests (Broadway et al., “Regulatory Mechanisms of Tryptic Inhibitory Activity in Cabbage Plants,” Phytochem. 29:3721-3725 (1990); Broadway et al., “Influence of Cabbage Proteinase Inhibitors in situ on the Growth of Larval Trichoplusia ni and Pieris rapae,” J.Chem. Ecol. 18:1009-1023 (1992)). In addition, cabbage foliar extracts containing PIs have been shown to significantly reduced growth and development of larval Lepidoptera and plant pathogenic fungi (Broadway, “Are Insects Resistant to Plant Proteinase Inhibitors?” J. Insect Physiol. 41:107-116 (1995); Broadway et al., “Influence of Cabbage Proteinase Inhibitors in situ on the Growth of Larval Trichoplusia ni and Pieris rapae,” J. Chem. Ecol. 18:1009-1023 (1992); Lorito et al., “Proteinase Inhibitors from Plants as a Novel Class of Fungicides,” Mol. Plant-Microbe Interact. 4:525-527 (1994)).
Genetic engineering of plants, which entails the isolation and manipulation of genetic material (usually in the form of DNA or RNA), and the subsequent introduction of that genetic material into plants or plant cells, offers considerable promise as a tool for the control of plant pests. If transgenic plants can be developed which express naturally occurring pest inhibitors, the need for expensive and potentially harmful chemical pest control measures is reduced. What is needed is a method of providing, and/or enhancing protection against herbivorous insects through the expression of the cabbage PI in crop plants.
The present invention is directed to overcoming these and other deficiencies in the art.